This invention relates to fluorescence and absorption spectroscopy using solid state standard coatings on optical glass or quartz. More specifically it relates to the calibration of fluorescence or absorbance reading microplate readers or spectrometers using a solid state device invention which is shaped to fit into square or bullet shaped microplate holders or spectrometer chambers and are read on appropriate fluorescence or absorption readers.
Standards which validate true capacity and integrity of various measuring devices are well known in the art. Whether standards used are solid, gas or liquid samples, they are quite common among all testing machines. The purpose of standards is to make sure that testing equipment is reading accurately, so that the measurements obtained on unknown quantities can be accepted as true and reliable. For this reason all testing machines have some standard that uniformly performs calibrations to assure readings which are consistent with the samples used and the unknowns tested. Traditionally this has required many samples of the materials at various compositions used as controls.
Spectroscopy is used to identify various unknown substances by reading spectroscopic patterns. Usually samples are tested over a wide range of wavelengths, from the Ultra Violet to Visible to Infra-Red bands of the spectrum. Testing relies on the consistent absorption or fluorescence by various compounds at specific wavelengths of light which produce a consistent pattern identifying the substance. Sometimes making an accurate identification of a substance is difficult because it is entirely dependent on absorption values of the substance. With fluorescent spectroscopy the user can examine the absorption or excitation of the compound as well as its emission of energy in the form of light as it returns to the ground state. For these substances, there are now two readings, which make it possible to identify unknowns with greater precision than ever before. To produce more accurate readings, solid state standards in microplates of any number can be made that standardize testing so that readings can be relied upon quickly and uniformly. The compounds absorb light during excitation and emit light of longer wavelength during emission. Furthermore, fluorescence spectroscopy is much more sensitive than U.V., visible or infra-red spectroscopy. This is because fluorescence is the excitation of the compound to a glow. This fluorescent glow can also be amplified to extremes by increasing voltage to the photomultiplier tube.
A review of the patent literature shows that the use of solid state standards with coatings in the calibration of microplate readers is a novel idea and not covered in the patent literature. For example, U.S. Pat. No. 4,661,711 uses an internal standard consisting of a fiberoptic bundle which fluoresces to calibrate a detector after splitting a light beam. The standard presented herein is an external optical glass moiety shaped to fit within a microplate holder or spectrometer chamber which is read in the corresponding instrument. The coated insert when read, gives a non changing reading provided that the photomultiplier (detector), voltage to the photomultiplier tube, lamp light output, monochomator if present and internal electronics do not change over time.
U.S. Pat. No. 5,414,258 entitled xe2x80x9cApparatus and method for calibration of fluorescencexe2x80x9d, describes an apparatus for reading the non visible fluorescence intensity of bar code shaped fluorescent targets which can be adjusted in the apparatus by changing the distance between the target and the detector or by changing the area of the target exposed to the detector. The solid state standards of the present invention can be used in absorption spectroscopy as well as fluorescence spectroscopy, and are shaped to fit microplate wells not as bar codes. The distance to the photomultiplier tubes is constant when reading these solid state standards in microplate readers although voltages applied to the photomultiplier tube (gain), will decrease or increase the relative magnitude of the fluorescence detected.
U.S. Pat. No. 3,854,050 entitled xe2x80x9cHigh precision fluorometer for measuring enzymatic substrates in tissuexe2x80x9d uses a fluorescent glass in combination with attenuating filters in a custom cuvette standard. The present invention however describes a microplate reader which uses absorbance or fluorescence standards on coated glass or quartz contoured to fit in a microplate or spectrometer; in contrast to the macro-cuvette holding apparatus described in the ""050 patent which employs a rectangular cuvette shaped reference. Instead of using attenuating filters to delimit the fluorescent emission of a specific reference standard, the invention disclosed herein uses industrial coatings which absorb in the Ultra Violet, Visible or Infra-Red electromagnetic ranges and are stable over time.
U.S. Pat. No. 5,503,910, entitled xe2x80x9cOrganic electroluminescence devicexe2x80x9d, describes an organic electroluminescence device consisting of a transparent anode (negatively charged electrode) which is coated with two layers of organic. When an electric field is applied, the first organic layer emits light at 380-480 nm, the second layer emits light at 480-580 nm and an organic in the first and second layer emits light at 580-620 nm. Overall the effect of this device is the emission of high energy white light. Alternatively in the present invention the reference standards emit monochromatic light when exposed to light delivered through an excitation or absorbance filter and are not dependant upon an electric field to emit light. More tellingly the samples or standards are coated with fluorescent or absorptive substances rather than an anode.
U.S. Pat. No. 4,868,126 entitled xe2x80x9cMethod of calibrating a fluorescent microscope using fluorescent calibration microbeads simulating stained cellsxe2x80x9d uses a hydrophilic microbead which covalently binds to a fluorescent molecule and can be visualized under a fluorescence microscope. The beads are microscopic (ranging from 1-20 microns) and therefore scatter light, which may be a limiting factor in a quantitative measurement device such as a microplate reader. Another problem with the microbeads-fluorescent molecule covalent bond is their lack of stability in solution. The coatings in the present invention are baked at 250 degrees Centigrade for several days by spectral coating experts resulting in a stable microwell insert.
U.S. Pat. No. 5,689,110 entitled xe2x80x9cCalibration method and apparatus for optical scannerxe2x80x9d, uses a beam splitter in a fluorescence spectrometer to compare two internal solid state standards, a calibration ruby and a gold standard. Neither of these standards are among the coatings which are utilized by the invention disclosed herein and moreover the standards are external to the measurement device. Furthermore a ratio method similar to the one described by the ""110 patent could not be used in a microplate reader.
U.S. Pat. No. 4,925,629 discloses a diagnostic device for preparing a standard calibration curve which employs a set of liquid standard tubes of diluted liquids which serve as a reservoir of connecting tubes. A micropipetting device then simultaneously draws up solution from eight tubes for serial transfer to other tubes. In contrast to the use of liquid as a standard, the present invention uses microplate pellets independently placed within the microplate and may be coated with known fluorescent or calorimetric substances. Another difference between the present invention and that of the ""169 patent is that due to evaporation, standards in a liquid state will decrease in volume and become more concentrated over time producing erroneous readings. The standards described herein are permanently fashioned to have a fixed pathlength. The microplate pellets disclosed herein are independently absorbing or fluorescent pieces so that they may be read and reread without further dilutional manipulation or transfer between plates.
Patent abstracts of Japan 01-142440 entitled xe2x80x9cCuvette holder for automated chemical analysisxe2x80x9d discloses the use of colored glass filters to calibrate a spectrophotometer. The solid state standards of the present invention are optical glass but are coated with absorbance or fluorescent constituents and is not simply using colored glass filters for calibration.
Patent abstracts of Japan No. 07-10594 entitled xe2x80x9cOptical Glass Filter for Calibrating Transmisivity or Absorbancexe2x80x9d describes the use of optical glass for calibration purposes. These band pass filters are composed of S10 less than SB greater than 2/SB greater than , alkali metal oxide and doping agents to vary the composition of the glass. The solid state microplate pellets of the present invention are optical glass but are coated with absorbance or fluorescent constituents which are not acting as band pass filters even though both inventions may read in the 300-700 nm range.
Patent abstracts of Japan No. 55-129728 employs a polished glass cell for use in a turbometric measuring devise. Although optical glass is used as a measuring cell, these polished cells are not or could not be used as a microplate insert and are not intended to be read in an absorbance or fluorescence type reader.
U.S. Pat. No. 5,582,168 describes the general measurement of fluorescence or turbidity in human tissue. Reflectance or fluorescence using a reflection of electromagnetic information is fundamentally different from measuring microsamples in an absorbance or fluorescence microplate reader. Moreover the ""168 patent does not use glass microplate pellets in its examination of the lens of the eye.
U.S. Pat. No. 4,971,439 entitled xe2x80x9cThe Wavelength Calibration Method and Apparatusxe2x80x9d is discussed next. The setting of the monochromator to obtain xe2x80x98zero order lightxe2x80x99 is done using a didyminium glass filter. As noted in the patent the first absorption of the glass filter occurs at a wavelength of 585.5 nm. The optical glass used in the present invention""s microplate pellets are not didyminium. Although microplate readers may have monochromators, this invention checks the monochromator as well as the excitation lamp and the photomultiplier tube by giving a constantly absorbing coating on the microplate pellet.
U.S. Pat. No. 4,135,816 entitled xe2x80x9cMethod and Application for Determining the Total Protein Content or Individual Amino Acidsxe2x80x9d uses a fluorometric method to examine the fluorescence and autofluorescence of a semi-solid suspension of protein and amino acids. We use coatings on microplate pellets to standardize the absorbance or fluorescence readings of microplate readers.
Accordingly it is desirable to have a method and apparatus for the calibration of fluorescence or absorbance reading microplate readers or spectrometers using a solid state device which is shaped to fit into square or bullet shaped microplate holders or spectrometer chambers and are read on appropriate fluorescence or absorption readers.
Infra-red and fluorescence microscopy employ analysis of the electromagnetic spectrum by using an excitation source such as a lamp, (deuterium, xenon, quartz, halogen or infra-red) that excites compounds to an excited state followed by their return to the original ground state. This condition allows for two readings in fluorescence measurement as opposed to one reading in the spectroscopic analysis. The return to the ground state in U.V., visible and infra-red is instantaneous (1xc3x9710xe2x88x9220 to 1xc3x9710xe2x88x9217 sec), while with fluorescence it takes a circuitous route before returning to the ground state (1xc3x9710xe2x88x9214 to 1xc3x9710xe2x88x929 sec). Emission of energy to the ground state is part of the dualistic nature of fluorescent compounds. Fluorescent compounds show both an excitation and emission spectrum absorbing light during excitation and emitting light of a longer wavelength, (ie. less energetic) light, during emission. This ability to have two readings allows for more accurate and precise readings of compounds than heretofore possible. The spectroscopic reading not being as precise as the fluorescent reading would be aided by having the latter test.
The present invention provides inserts of solid state compounds which intrinsically fluoresce or absorb at a given wave length or have a coating which fluoresces or absorbs at a given wavelength when placed in the reading compartment of a given fluorescence or absorption reader. The inserts are fashioned into the desired shape and are coated by a process which includes baking the coated pieces at 250 degree centigrade for various periods of time (over several days). The coating solutions are chosen to produce various wavelength readings when read upon the coated pieces. Coatings include AgBr, AgCl, Al2O3, BaF2, CaF2, CdTe, CsI, Ge, KBr, KCl, KRS-5, Si, NaCl, Si, SiO2, TiO2, ZnS, ZnSe, HFO2, MgO, Fluroisothiocyanate (FITC), Fluorescene, Rhodamine B, Quinine Sulfate, Bodipy and Green Fluorescent Protein. These coated insert are durable, and provide reliable readings over time (over 3 years). The fluorescence and absorbance readings will not to shift 0.1 OD (Optical Density) in absorbance units and in fluorescence units not more than 1000 fluorescence units within the full scale of 75,000 fluorescence units. The inserts can be fashioned to read at any Optical Density (hereinafter xe2x80x9cODxe2x80x9d) reading so that a set of inserts with ascending OD units could be used as a concentration curve when read on the appropriate reader.
Furthermore when using a set of solid state standards to calibrate a run, an additional insert with wavelength readings identical to one of the set of calibration standards could be included to check whether the reading one is getting is still within the limits of the assay as described by the calibration curve. Also the insert could be used in UV-visible, Raman, infra-red, and FTIR (fourier transformation infra-red),laser spectroscopy and luminescence spectroscopy. The fluorescence insert can be used as a light source for a luminometer by placing the insert in sunlight for fifteen minutes then immediately placing the insert still in its microplate within the luminometer. The insert will autofluoresce and decay in intensity upon reading in the luminometer. In addition cuvette shaped coated calibration pieces can be used in spectrophotometers and spectrophotometers to calibrate monochromators.
This invention discloses a calibration standard which is unwavering in optical density or relative fluorescence units, stable over time (not changing in reading more than 0.1 Optical Density units in instruments from 0-3.0 Optical Density units or more than 1000 fluorescent units in instruments measuring up to 75,000 Relative Fluorescence units over a 3 year period).
These and other advantages of the present invention will become more thoroughly apparent through the following description of the preferred embodiments and the accompanying drawings.